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Overview of Cell Lysis and Protein Extraction

Cell lysis is the first step in cell fractionation, organelle isolation and protein extraction and purification. As such, cell lysis opens the door to a myriad of proteomics research methods. Many techniques have been developed and used to obtain the best possible yield and purity for different species of organisms, sample types (cells or tissue) and target molecule or subcellular structure.

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Protein Methods Library Home

Structure and Diversity of Cells

All cells have a plasma membrane, a protein-lipid bilayer that forms a barrier separating cell contents from the extracellular environment. Lipids comprising the plasma membrane are amphipathic, having hydrophilic and hydrophobic moieties that associate spontaneously to form a closed bimolecular sheet. Membrane proteins are embedded in the lipid bilayer, held in place by one or more domains spanning the hydrophobic core. In addition, peripheral proteins bind the inner or outer surface of the bilayer through interactions with integral membrane proteins or with polar lipid head groups. The nature of the lipid and protein content varies with cell type and species of organism.

Plasma membrane lipid bilayer structure. Cell membrane structure. Illustration of a lipid bilayer comprising outer plasma membrane of a cell.

In animal cells, the plasma membrane is the only barrier separating cell contents from the environment, but in plants and bacteria the plasma membrane is also surrounded by a rigid cell wall. Bacterial cell walls are composed of peptidoglycan. Yeast cell walls are composed of two layers of ß-glucan, the inner layer being insoluble to alkaline conditions. Both of these are surrounded by an outer glycoprotein layer rich in the carbohydrate mannan. Plant cell walls consist of multiple layers of cellulose. These types of extracellular barrier confer shape and rigidity to the cells. Plant cell walls are particularly strong, making them very difficult to disrupt mechanically or chemically. Until recently, efficient lysis of yeast cells required mechanical disruption using glass beads, whereas bacterial cell walls are the easiest to break compared to these other cell types. The lack of an extracellular wall in animal cells makes them relatively easy to lyse.

Related literature...
Cell Lysis Technical Handbook.
Cell Lysis Technical Handbook

Cell Lysis and Protein Extraction

Historically, physical lysis was the method of choice for cell disruption and extraction of cellular contents; however, it often requires expensive, cumbersome equipment and involves protocols that can be difficult to repeat due to variability in the apparatus (such as loose-fitting compared with tight-fitting homogenization pestles). Also, traditional physical disruption methods are not conducive for high throughput and smaller volumes typical of modern laboratory research.

In recent years, detergent-based lysis methods have become the norm. Through empirical testing by trial and error, different detergent-based solutions composed of particular types and concentrations of detergents, buffers, salts and reducing agents have been developed to provide the best possible results for particular species and types of cells. Detergents have both lysing and solubilizing effects.

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Traditional Cell Lysis Methods

Detergents for Cell Lysis

Cell Lysis Solutions

Cell Fractionation and Organelle Isolation

By careful optimization of physical disruption techniques, detergent-buffer solutions and density gradient methods, procedures have been developed to enable separation of subcellular structures or classes of compounds. For example, with the appropriate detergents, hydrophobic membrane proteins can be solubilized and separated from hydrophilic proteins. A combination of tools and steps enable intact nuclei, mitochondria and other organelles to be isolated for study or protein solubilization.

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Cell Fractionation and
Organelle Enrichment

Protease Inhibition and Protein Stabilization

Cell lysis disturbs the carefully controlled cellular environment, allowing endogenous proteases and phosphatases to become unregulated. As a result extracted proteins become degraded or artifactually modified by the activities of these molecules.

To prevent these effects and obtain the best possible protein yield in cell lysis, protease and phosphatase inhibitors are added to the lysis reagents. Numerous compounds have been identified and used to inactivate or block the activities of proteases and phosphatases by reversibly or irreversibly binding to them.

When the goal of cell lysis is to purify or test the function of a particular protein, special attention must be given to the effects of the lysis reagents on the stability and function of the protein(s) of interest. Certain detergents will inactivate the function of particular enzymes, and long-term stability of extracted/purified proteins often requires that they be removed from the initial lysis reagents and/or stabilized by addition of particular compounds.

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Protease and Phosphatase Inhibitors for Cell Lysis

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Inhibitor Cocktails

Protein Stabilizing Cocktail

Protein Refolding

Some cell lysis and protein solubilization methods cause the denaturation of proteins. Functional tests with such proteins requires that they be renatured. Many proteins spontaneously refold into their native, functional structures when the denaturing solubilization reagents are removed by dialysis. Other proteins, however, will fold into non-functional and even insoluble forms by this process. In such cases, specialized sets of buffer conditions must be tested to identify those that promote the highest possible yield of properly refolded protein.

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Protein Refolding Kit

Inclusion Body Solubilization Reagent

Written and/or reviewed by Douglas Hayworth, Ph.D.

Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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